Principal Investigators: GREALLY, J.M., LEVY, M. Project abstract IN VIVO IMAGING OF X INACTIVATION. We propose to develop a system for in vivo imaging of the epigenetic regulatory processes involved in X chromosome inactivation. X inactivation is a well-studied paradigm of epigenetic gene regulation, involving the silencing of the majority of the genes on one X chromosome in female cells, part of the process of dosage compensation in mammals. A number of epigenetic regulatory processes have been found to contribute to the inactivation process, which when imaged using immunofluorescence on fixed cells generate a signal throughout the chromosome territory of the inactive X. The robustness of this signal makes X inactivation an attractive system for the development of in vivo imaging approaches. The inactive X is characterized by the presence of repressive post-translational histone modifications such as histone H3 lysine 9 trimethylation (H3K9me3) and H3K27me3, modifications established by polycomb group proteins which, when mutated, are associated with the failure of X inactivation. There are, however, other regulatory mediators implicated with functions that are less obviously related to the establishment of these chromatin states, functions such as helicase activity, RNA-binding, matrix-attachment region DNA-binding, or those functions associated with chromosomal structural maintenance motifs. As a means of understanding how each component of the X inactivation system interacts functionally, an in vivo system would allow the observation of sequential localization of the protein mediators and histone modifications to the inactivating X chromosome, thus establishing a likely hierarchy of regulation in this complex epigenetic process. In order to develop such a system, a number of areas of expertise need to be assembled. The project starts with the in vitro generation of histone peptides (and eventually entire reconstituted nucleosomes) with methylation and ubiquitination marks (David Allis and Tom Muir, Rockefeller University) that are then used for in vitro selection by co-PI Matthew Levy (Einstein) to create RNA aptamers specifically binding to these post- translational modifications. These aptamers are then linked in an expression construct to RNA hairpins bound by fluorescently-tagged phage coat proteins, a system pioneered by co-investigator Robert Singer (Einstein) as a means of tracking RNA in vivo in transcription studies. This project represents the first use of the same system for epigenetic studies. The cell type in which the system will be optimized will be a female mouse embryonic stem cell line, allowing not only X inactivation studies but also the broader use of this system in pluripotent cells when made available to the scientific community. The X inactivation studies will be facilitated by the development of fluorescent tags for the candidate protein mediators of X inactivation (Edith Heard, Institut Curie, Paris, France). The project is thus based on a strong and multifaceted foundation of expertise and resources. PHS 398/2590 (Rev. 11/07) Continuation Format Page